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Is Gdh a Marker for Mitochondria in Brain? / James C Fordham University Masthead Logo DigitalResearch@Fordham Chemistry Faculty Publications Chemistry 1986 The ubs cellular localization of glutamate dehydrogenase (gdh): is gdh a marker for mitochondria in brain? / James C. K. Lai, Kwan-Fu Rex Sheu, Young Tai Kim, Donald D. Clarke, and John P. Blass Department of Neurology, Cornell University Medical College and Altschul Laboratory for Dementia Research Burke Rehabilitation Center White Plains, NY 10605 and Department of Medicine Cornell University Medical College New York, NY 10021 James C. K. Lai Cornell University. Department of Neurology and Neuroscience Kwan-Fu Rex Sheu Burke Rehabilitation Center Recommended Citation Lai, James C. K.; Sheu, Kwan-Fu Rex; Kim, Young Tai; and Clarke, Donald Dudley PhD, "The ubcs ellular localization of glutamate dehydrogenase (gdh): is gdh a marker for mitochondria in brain? / James C. K. Lai, Kwan-Fu Rex Sheu, Young Tai Kim, Donald D. Clarke, and John P. Blass Department of Neurology, Cornell University Medical College and Altschul Laboratory for Dementia Research Burke Rehabilitation Center White Plains, NY 10605 and Department of Medicine Cornell University Medical College New York, NY 10021" (1986). Chemistry Faculty Publications. 21. https://fordham.bepress.com/chem_facultypubs/21 This Article is brought to you for free and open access by the Chemistry at DigitalResearch@Fordham. It has been accepted for inclusion in Chemistry Faculty Publications by an authorized administrator of DigitalResearch@Fordham. For more information, please contact [email protected]. Young Tai Kim Cornell University. Medical College Donald Dudley Clarke PhD Fordham University, [email protected] Follow this and additional works at: https://fordham.bepress.com/chem_facultypubs Part of the Biochemistry Commons • Neurochemical Research, Vol. 11, No.5, 1986, pp. 733-744 THE SUBCELLULAR LOCALIZATION OF GLUTAMATE DEHYDROGENASE (GDH): Is GDH a Marker for Mitochondria in Brain? JAMES C. K. LAI, KwAN-Fu REx SHED, YouNG TAI KIM*, DoNALD D. CLARKE, and, JoHN P. BLAss Department of Neurology, Cornell University Medical College and Altschul Laboratory for Dementia Research Burke Rehabilitation Center White Plains, NY 10605 and *Department of Medicine Cornell University Medical College New York, NY 10021 Accepted December 3, 1985 Glutamate dehydrogenase (GDH, EC 1.4.1.2) has long been used as a marker for mitochondria in brain and other tissues, despite reports indicating that GDH is also present in nuclei of liver and dorsal root ganglia. To examine whether GDH can be used as a marker to differentiate between mitochondria and nuclei in the brain, we have measured GDH by enzymatic activity and on immunoblots in rat brain mitochondria and nuclei which were highly enriched by density-gradient centrifugation methods. The activity of GDH was enriched in the nuclear fraction as well as in the mitochondrial fraction, while the activities of other "mitochon­ drial" enzymes (fumarase, NAD-isocitrate dehydrogenase and pyruvate dehy­ drogenase complex) were enriched only in the mitochondrial fraction. lmmunob­ lots using polyclonal antibodies against bovine liver GDH confmned the presence of GDH in the rat brain nuclear and mitochondrial fractions. The GDH in these two subcellular fractions had a very similar molecular weight of 56,000 daltons. The mitochondrial and nuclear GDH differed, however, in their susceptibility to solubilization by detergents and salts. The mitochondrial GDH could be solubi- Please address reprint requests to: Dr. Sheu, Burke Rehabilitation Center, 785 Mamaroneck Avenue, White Plains, NY 10605. Dr. Lai's current address is: Department of Neurology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021. Dr. Clarke's current address is: Chemistry De­ partment, Fordham University, Bronx, NY 10458. 733 0364-3190/86/0500-0733$05.00/0 © 1986 Plenum Publishing Corporation 734 LAI ET AL. lized by extraction with low concentrations of detergents (0.1% Triton X-100 and 0.1% Lubrol PX), while the nuclear GDH could be solubilized only by elevated concentrations of detergents (0.3% each) plus KCl (> 150 mM). Our results indicate that GDH is present in both nuclei and mitochondria in rat brain. The notion that GDH may serve as a marker for mitochondria needs to be re-evaluated. INTRODUCTION Glutamate dehydrogenase [GDH, EC 1.4.1.2, L-glutamate: NAD oxido­ reductase (deaminating)] has long been thought to be confmed to mito­ chondria (1), and has been used extensively as a marker to identify mi­ tochondria in subcellular fractions of many tissues, including the brain. However, the inference that the GDH activity can be used as a measure of mitochondrial contamination in subcellular fractions other than mito­ chondria may not be justified. Di Prisco and his coworkers have purified GDH from isolated nuclei of bovine liver (2). This nuclear GDH was not due to a contamination of mitochondrial origin since the immunochem­ ical, catalytic and chromatographic properties of this nuclear GDH were not identical, although very similar, to those of mitochondrial GDH (2, 3). Kato and Lowry (4) have also found GDH activity in the nuclei dis­ sected from single dorsal root ganglion neurons of the rabbit. The GDH activity persisted in the trimmed and subdivided nucleus, and accounted for i the specific activity (on a dry weight basis) relative to the rest of the cell (4). The brain consists of many different types of cells. In accord with this, variation of GDH has been reported at the regional level (5, 6), and is likely to be manifested at the cellular and subcellular levels (7 -9). A significant amount of GDH confined to the nuclei may result in a mis­ judgment of the purity of isolated nuclear fractions if GDH is employed as a marker for mitochondria. In this study, we determined whether there is a significant amount of GDH in the isolated brain nuclei by using activity measurement and im­ munoblotting techniques. The results support the long overlooked notion that GDH is also present in nuclei, and that GDH may not be a proper marker for mitochondria, especially in brain tissue. Preliminary results of this study have been presented (10, 11). EXPERIMENTAL PROCEDURE Preparation of Nuclei. Four male Wistar rats (Charles River Breeding Laboratories, Sto­ neridge, NY) of 45-60 days old were used in each experiment. The forebrains were dissected out as described previously (12), the superficial blood vessels removed, and the brains rinsed once with the ice-cold isolation medium which contained: 0.32 M sucrose, 1 mM MgCh, • SUBCELLULAR LOCALIZATION OF GDH 735 and 1 mM 3-(N-morpholino)propanesulfonic acid, pH adjusted to 6.6 with Tris base [MOPS(Tris), pH 6.6]. The brains were then minced with scisssors, and further rinsed at least 3 times with the isolation medium. The chopped tissue was manually homogenized with 9-volumes of isolation medium in an all glass Dounce homogenizer with loose pestle (clearance: 0.007 inch) using 10 up- and down-strokes. The homogenate was filtered twice through 110 m stainless-steel meshes to remove cellular debris and capillaries. The filtrate was then centrifuged at 3,000 g for 3 min. The supernatant and the loose portion of the pellet were decanted into another centrifuge tube. The tightly packed pellet, which consisted mostly of capillaries, some cellular debris and portions of large nuclei, was discarded. The decanted material was then centrifuged at 3,000 g for 3 min. The pellet, which consisted mainly of nuclei, was resuspended in 30 ml of 2 M sucrose, 1 mM MgCh and 1 mM MOPS(Tris), pH 6.6, and divided into two 30 ml centrifuge tubes. To each tube, 15 ml of 1 M sucrose, 1 mM MgCh and 1 mM MOPS(Tris), pH 6.6 was carefully layered on top of the suspension which contained nuclei. The gradient was centrifuged at 110,000 g for 1 hr in a SW-27 rotor in a Beckman L5-50B ultracentrifuge. The nuclear pellet was then washed once with the isolation medium, suspended in the isolation medium (10 mgp/ml) and stored at - sooc in small aliquots. The yield was 5.9 + 0.9 mgp/g wet weight (mean + SD; n = 7). Preparation of Mitochondria and Cytosol. Male Wistar rats of similar ages (see above) were used in these preparations. Non-synaptic mitochondria from forebrains were isolated by the Ficoll-sucrose density gradient method, as detailed previously (13). The postmito­ chondrial supernatant was further centrifuged at 80,000 g for 90 min to obtain the cytosolic fraction. The homogenate, cytosol and mitochondria [resuspended 10 mgp/ml in 0.32 M sucrose and 5 mM N-2-hydroxyethylpiperazine-N' -2' -ethanesulfonic acid, pH adjusted to 7.4 with KOH] were also stored at - 80°C in small aliquots. Enzyme ~ctivity Measurement. The GDH activity was determined by the 2-oxoglutarate-, NHt -, and ADP-dependent NADH oxidation, as described by Lai and Clark (14). The pyruvate dehydrogenase complex (PDHC, EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) was measured according to Sheu et al. (15). The NAD-linked isocitrate dehydrogenase (ICDH, EC 1.1.1.41) and fumarase (FUM, EC 4.2.1.2) were measured according to Lai and Clark (14). These enzyme activities were measured within a time period when they remained fully active: the ICDH and PDHC were assayed within 2 days, and the GDH and fumarase within a week. Antibodies Against GDH (Anti-GDH). The bovine liver GDH obtained from Sigma Chem­ ical Co. (St. Louis, MO) was further purified by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The GDH was electrophorased in 7.5% polyacrylamide slab gels (Hoefer 500SE unit) according to Weber and Osborn (16), and briefly stained with Coomassie blue and destained. The GDH band was cut out from the gel slabs, and embedded with 6% polyacrylamide gel in the second 1.5 em gel tubes.
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